Uncoupling protein (UCP) is a proton transporter present in the mitochondrial inner membrane. Since UCP changes intracellular energy stored as fat to heat without using other energy consuming processes, UCP is considered to play an important role in maintenance of body temperature in homeothermal animals. Because of this function, UCP is considered to be an important factor that determines the efficiency of energy metabolism in homeothermal animals.
Three molecular species of uncoupling proteins have been identified to date, and are called uncoupling proteins-1 (UCP-1), -2 (UCP-2 or UCPH), and -3 (UCP-3).
UCP-1, the first isolated among the uncoupling protein family, is specifically expressed in brown fat cells (Line, C. S. and Klingerberg, M. (1980), FEBS Lett., 113, 299-303; Jacobsson, A. et al. (1985), J. Biol. Chem., 260, 16250-16254; Bouillaud, F. et al. (1986), J. Biol. Chem., 261, 1487-1490). UCP-2 was isolated as a homologue of UCP-1, and confirmed to be widely expressed in various organs (Gimeno, R. E. et al. (1997), Diabetes, Vol. 46, 900-906; Fleury, C., et al. (1997), Nature Genet., Vol. 15, 269-272). UPC-3 was isolated as a UCP specifically expressed in muscles (Vidal-Puig, A. et al. (1997), Biochem. Biophys. Res. Commun., Vol. 235, No. 1, 79-82; Boss, O. et al. (1997), FEBS Lett., 408, 33-38).
Generally, UCP-1 is considered to play an important role in maintenance of body temperature in rodents and hibernants. Basically, the number of brown fat cells that mainly express UCP-1 is lower in large sized animals and animal species inhabiting relatively warm climates (Rothwell, N. J. and Stock, M. J. (1979), Nature, Vol. 281, 31-35). Thus, in these animals including humans, UCP-2 or UCP-3, not UCP-1, may mainly be responsible for the control of the normal body temperature maintenance system and energy consuming process (Hosoda, K. et al. (1998), Obesity Research (Himan Kenkyu), Vol. 4, No. 3, 31-35; Enerback, S. et al. (1997), Nature, Vol. 387, 90-93).
Therefore, it may be possible to adjust the energy consumption/accumulation balance by controlling the gene expression or activity of UCP-2 or UCP-3 in these animals including humans (Hosoda, K. et al. (1998), Obesity Research (Himan Kenkyu), Vol. 4, No. 3, 31-35; Enerback, S. et al. (1997), Nature, Vol. 387, 90-93). In humans, enhancement of energy consumption is considered to promote consumption of not only dietary energy but also energy accumulated as fat. Accordingly, a decrease of body fat in humans may lead to improvement of obesity, the major cause of lifestyle diseases which become a problem in developed countries in recent years (Fleury, C. et al. (1997), Nature Genetics, Vol. 15, 269-272).
UCP-2 is also considered to be the major cause of fever observed in immunological inflammation such as infection, and inhibition of UCP-2 gene activity may reduce fever in immunological inflammation (Shigenaga, F. R. et al. (1998), Biochiem. Biophys. Res. Commun., Vol. 244, No. 1, 75-78).
In animals, especially in higher animals, organs differentiate and mature upon biogenesis, and develop to exert various functions. During this process, various organ-specific proteins are transiently or constantly expressed and provide the organ-specificity.
The general gene expression control system in animals includes the transcription induction system (promoter, enhancer). Promoter regions are generally located adjacent to the 5′ upstream region of base sequences on chromosomes that are normally transcribed into messenger RNAs. Transcriptional regulatory protein is bound to or dissociated from a base sequence generally called the regulatory sequence in promoter regions, by which the transcription level of genes located downstream of the 3′ region is regulated. Therefore, the transcriptional gene expression level can be estimated from the promoter activity to some extent. It is also known that the base sequences located downstream of the 3′ region of a promoter do not affect the promoter activity in most cases. Therefore, promoter activity can be readily measured by substituting the transcribed messenger RNA for a base sequence encoding a protein with detectable enzyme activity (reporter). Recent technical innovation has made measurement of promoter activity using reporters very sensitive and simple, and measurement of promoter activity is used in drug screening and analysis of biological function.
For example, transcriptional regulatory factors of fat cell differentiation include peroxisome proliferation-activated receptor γ (PPAR γ) (Tontontz, P. et al. (1995), Curr. Opin. Genet. Dev., Vol. 5, 571-576), retinoid X receptor (RXR), CCAAT/enhancer binding protein (C/EBP) (Cornelius, P., et al. (1994), Annu. Rev. Nutr. Vol. 14, 99-129), etc. The transcriptional regulation by these factors is closely involved in the gene expression related to fat cells. It has been reported that the promoter regions of fat cell-related genes including UCP-2 gene contain the binding sequences for these transcriptional regulatory factors (regulatory sequences). These sequences in promoters are considered to play important roles in the actual regulation of UCP-2 transcription in vivo.
Accordingly, substances that enhance expression of UCP-2 or UCP-3 gene and protein may be used as anti-obesity drugs that reduce body fat content. UCP-2 is also considered to be the major cause of fever in immunological inflammation observed in infection, and substances that inhibit UCP-2 gene activity may reduce fever in immunological inflammation.
If a cell line expressing an appropriate reporter gene connected to the promoter region described above is established, the cell line may be used for screening a drug that promotes or inhibits the UCP-2 expression. In screening substances that may be used as anti-obesity drugs, responses more similar to those in vivo can be obtained by including these regulatory sequences in the promoter-reporter system, which is very advantageous in screening human anti-obesity drugs.
However, human UCP-2 promoter containing the regulatory sequence had not yet been identified, and no simple screening method using the promoter described above had been available for substances that affect the human UCP-2 gene expression.